Alphaviruses are an important cause of mosquito-borne encephalitis. Our overall aim is to determine the mechanism of alphavirus transcription and to understand how the synthesis of viral minus and plus strand RNA is regulated. The viral encoded nonstructural proteins of the alphaviruses Sindbis virus (SIN) and Semliki Forest virus (SFV) which initiate viral replication by transcribing the infecting viral plus-strand genome RNA into a complementary minus-strand RNA will be characterized as to their function in viral replication. Temperature sensitive mutants of SIN and SFV and their revertants that we have characterized previously will be used to identify the functions of the viral proteins that are required for minus-strand RNA synthesis and to determine if the regulation of minus-strand RNA synthesis involves cis-acting or trans-acting viral functions. We will study defective interferring particles (DIs) of SIN HR to determine whether nondefective virus is capable of shutting off the synthesis of the minus-strand template for the DI RNA. We will sequence, by the method of primer extension using reverse transcriptase and dideoxynucleotides with purified viral genomes as templates, the genes encoding the third and fourth nonstructural proteins of SIN HR, ts6, ts11 and ts24 and revertants of these mutants. Our previous research leads us to suspect that the third or the fourth nonstructural protein of the alphavirus genome is encoded by the B or the F cistron. We will continue to develop an in vitro assay to characterize alphavirus transcription, particularly, minus strand synthesis using viral RNA produced in vitro from cloned viral RNA. We plan to clone the nonstructural genes and required flanking sequences of both SIN HR and selected temperature sensitive mutants of SIN HR into Riboprobe Gemini plasmid vectors in order to analyze the sequence requirements for synthesis and regulation of alphavirus plus-strand and minus-strand RNA synthesis. Lastly, we will identify and characterize the viral encoded protease that processes the polyprotein precursor to the viral nonstructural proteins and that appears to be temperature sensitive in SIN HR ts17.